Structure and Function of Protein C

描述（由申请人提供）：这一竞争性更新申请的重点是活化蛋白C （APC）的功能蛋白质组学研究，这种酶具有两种主要的、不同的活性——(1)在需要APC辅助因子的反应中，通过靶向凝血因子Va和Vllla来发挥抗凝活性；(2)通过靶向两种APC受体，蛋白酶活化受体1 （PAR1）和内皮蛋白C受体（EPCR），对细胞产生直接的抗凋亡和抗炎作用。我们已发表的工作和未发表的初步数据直接导致了我们提出的研究，并为我们的假设提供了有力的支持。我们假设APC对细胞的epcr依赖性作用至少部分来自于APC在PAR1中Arg41位点的裂解，该反应涉及PAR1特异性外源位点APC与Va外源位点不同。我们假设细胞上的PAR1通过细胞外蛋白环和/或通过跨膜螺旋-螺旋相互作用直接与EPCR相互作用。我们假设有其他辅助因子或接头影响APC对细胞的直接作用。我们假设APC的抗凋亡活性需要胞吞作用，并且胞吞后的APC直接在细胞内发挥抗凋亡活性。我们假设APC通过下调caspase 3的生成来发挥抗凋亡活性，并且APC可能在线粒体释放cyt c的上游起作用。为了验证这些假设，我们提出：1)扩大突变框架，以了解APC对其关键底物的酶靶向作用；2)表征高密度脂蛋白（HDL）和因子V作为APC辅助因子；3)阐明APC直接作用于内皮细胞的机制；4)评估APC新的细胞内抗凋亡活性。在相关的转化研究中，我们建议评估血栓性疾病与APC受体、PAR1和EPCR之间的关系，以及血栓性疾病与APC脂质辅助因子、糖基神经酰胺血浆水平之间的关系。这些研究将提高我们理解、诊断和治疗血栓性疾病的能力。

DESCRIPTION (provided by applicant): This competing renewal application is focused on functional proteomics studies of activated protein C (APC), an enzyme that exerts two major, distinct activities - (1) anticoagulant activity by targeting coagulation factors Va and Vllla in reactions requiring APC cofactors and (2) direct anti-apoptotic and anti-inflammatory effects on cells by targeting two APC receptors, protease activated receptor 1 (PAR1) and Endothelial Protein C Receptor (EPCR). Our published work and unpublished preliminary data lead directly to the proposed studies and provide strong support for our hypotheses. We hypothesize that EPCR-dependent effects of APC on cells derive, at least in part, from APC's cleavage at Arg41 in PAR1 in a reaction involving PAR1-specific exosites on APC that differ from factor Va exosites. We hypothesize that PAR1 on cells interacts directly with EPCR via extracellular protein loops and/or via transmembrane helix-helix interactions. We hypothesize that there are additional cofactors or adaptors that influence APC's direct effects on cells. We hypothesize that APC endocytosis is required for APC's anti-apoptotic activity and that endocytosed APC directly exerts intracellular anti-apoptotic activity in cells. We hypothesize that APC exerts antiapoptotic activity by downregulating caspase 3 generation and that APC might act upstream of mitochondrial release of cyt c. In testing these hypotheses, we propose: 1) to expand the mutational framework for understanding APC's enzymatic targeting of its key substrates, 2) to characterize high density lipoprotein (HDL) and factor V as APC cofactors, 3) to clarify mechanisms responsible for APC's direct effects on endothelial cells, and 4) to evaluate APC's novel intracellular anti-apoptotic activity. In related translational research, we propose to assess relationships between thrombotic disease and the APC receptors, PAR1 and EPCR, and between thrombotic disease and plasma levels of the APC lipid cofactor, glucosylceramide. These studies will improve our ability to understand, diagnose and treat thrombotic diseases.